The haemostatic balance is dependent on interactions between platelets, vessel-wall coagulation and fibrinolysis. Upon vessel wall injury platelets adhere to the subendothelium and form aggregates. Next, coagulation is initiated ultimately resulting in the production of fibrin fibers that stabilize the platelet plug. All these steps are important to form a stable blood clot that is resistant against the rheological forces of the blood flow. Thrombin is a key player in these processes as it is responsible for a whole concert of reactions, for instance it solidifies the blood by formation of fibrin, activates platelets, activates positive feedback mechanisms and interacts with the endothelial receptors to start negative feedback mechanisms. Activated platelets, in their turn catalyze thrombin formation. Thrombin has a short half-life, approximately a few minutes, which is caused by the binding of thrombin inhibitors like antithrombin. Antithrombins also act as regulatory proteins, which can inactivate the first traces of thrombin before they can augment thrombin generation by positive feedback reactions. This prevents systemic clot formation. Fibrinolysis on its turn solubilizes the fibrin containing platelet plug. Disequilibrium of this haemostatic balance in one or more compartments (vessel wall, blood cells, coagulation and fibrinolysis) may result in a thrombotic response or hemorrhagic phenotype which even can be life threatening.
When abnormalities of the haemostatic system occur, it is essential to diagnose, monitor, and manage the patient in order to optimize therapeutic intervention.
Known haemostasis assays involve end-point assays, which detect the clotting time of blood plasma (clotting assays) or real-time clot-lysis by means of turbidimetry (fibrinolysis assays). Although performed routinely, the currently available coagulation assays have inherent limitations that make them potentially unreliable as tools for monitoring increased coagulation. Moreover, there is not always a good correlation between the results of coagulation tests and the prevention of postoperative hemorrhage or recurrent thrombosis (Hemker et al. Curr. Opin. in Hematology 2004, 11:170-175).
Most of the limitations relate to the fact that these are end-point tests that measure the time of clot formation in vitro and require the addition of exogenous reagents (such as tissue factor, kaolin and Ca2+ ions to replenish those bound by an anticoagulant), and thus do not necessarily reflect the patient's thrombotic potential (clotting potential).
As compared to the tests described above, EP 420 332 discloses an improved thrombin generation assay. In this assay not only information is gathered about the clotting of plasma but also about the total thrombin generation after clot formation. These assays were first performed with chromogenic substrates and later on with fluorogenic substrates. Furthermore, several thrombin generation assays with platelet-poor and platelet-rich plasma are disclosed.
The chromophores (e.g. p-nitro-anilin [p-NA]) used in chromogenic assays are typically assessed using a wavelength of 405 nm. An important drawback of chromogenic substrates is that both fibrin and platelets in aqueous solutions interfere with the assessment of chromogenic substrates at 405 nm, and that the measurement as such is unreliable. Therefore, the use of fluorogenic substrates is nowadays more popular. Fluorogenic substrates are analogous to chromogenic substrates. The difference is that upon enzymatic action the substrates release a group which can be determined with high sensitivity using a fluorometer. Fluorometric assessment of thrombin generation further has the advantage that fibrin or platelets do not interfere with the analysis.
Furthermore, the use of multiple fluorogenic substrates with different characteristics allow the detection of several products in one sample as described in WO 2006/072602. The use of fluorogenic substrates also has significant disadvantages: the standard laboratory equipment used for analysis of the coagulation system does not support fluorometric analysis. Thus the analysis requires additional instrumentation (fluorometer). In addition the trend of the last decade has been to implement all coagulation tests wherever possible on one analyzer in order to simplify the testing procedure and minimize labor costs. The use of a separate instrument for measuring thrombin generation significantly reduces its applicability as a routine method. Further, the fluorescent signal has the drawback of not being linear with product concentration. A need exists for a new assay for measuring thrombin generation and generation of other blood clotting factors that does not have the above indicated drawbacks, that is simpler and can measure the generation of blood clotting and fibrinolytic factors such as thrombin and plasmin in a direct manner, preferably in a linear mode. It is an object of the present invention to provide such assay.